This invention relates generally to the field of cell biology of embryonic cells, and the molecular biology of promoter controlled viral vectors. More specifically, it describes a technology for removing undifferentiated cells from populations derived from pluripotent stem cells using selectively expressed lytic vectors.
Precursor cells have become a central interest in medical research. Many tissues in the body have a back-up reservoir of precursors that can replace cells that are senescent or damaged by injury or disease. Considerable effort has been made recently to isolate precursors of a number of different tissues for use in regenerative medicine.
U.S. Pat. No. 5,750,397 (Tsukamoto et al., Systemix) reports isolation and growth of human hematopoietic stem cells which are Thy-1+, CD34+, and capable of differentiation into lymphoid, erythroid, and myelomonocytic lineages. U.S. Pat. No. 5,736,396 (Bruder et al.) reports methods for lineage-directed differentiation of isolated human mesenchymal stem cells, using an appropriate bioactive factor. The derived cells can then be introduced into a host for mesenchymal tissue regeneration or repair.
U.S. Pat. No. 5,716,411 (Orgill et al.) proposes regenerating skin at the site of a burn or wound, using an epithelial autograft. U.S. Pat. No. 5,766,948 (F. Gage) reports a method for producing neuroblasts from animal brain tissue. U.S. Pat. No. 5,672,499 (Anderson et al.) reports obtaining neural crest stem cells from embryonic tissue. U.S. Pat. No. 5,851,832 (Weiss et al., Neurospheres) reports isolation of putative neural stem cells from 8-12 week old human fetuses. U.S. Pat. No. 5,968,829 (M. Carpenter) reports human neural stem cells derived from primary central nervous system tissue.
U.S. Pat. No. 5,082,670 (F. Gage) reports a method for grafting genetically modified cells to treat defects, disease or damage of the central nervous system. Auerbach et al. (Eur. J. Neurosci. 12:1696, 2000) report that multipotential CNS cells implanted into animal brains form electrically active and functionally connected neurons. Brustle et al. (Science 285:754, 1999) report that precursor cells derived from embryonic stem cells interact with host neurons and efficiently myelinate axons in the brain and spinal cord.
Considerable interest has been generated by the development of embryonic stem cells, which are thought to have the potential to differentiate into many cell types. Early work on embryonic stem cells was done in mice. Mouse stem cells can be isolated from both early embryonic cells and germinal tissue. Desirable characteristics of pluripotent stem cells are that they be capable of proliferation in vitro in an undifferentiated state, retain a normal karyotype, and retain the potential to differentiate to derivatives of all three embryonic germ layers (endoderm, mesoderm, and ectoderm).
Development of human pluripotent stem cell preparations is considerably less advanced than work with mouse cells. Thomson et al. propagated pluripotent stem cells from lower primates (U.S. Pat. No. 5,843,780; Proc. Natl. Acad. Sci. USA 92:7844, 1995), and then from humans (Science 282:114, 1998). Gearhart and coworkers derived human embryonic germ (hEG) cell lines from fetal gonadal tissue (Shamblott et al., Proc. Natl. Acad. Sci. USA 95:13726, 1998; and U.S. Pat. No. 6,090,622).
Both hES and hEG cells have the long-sought characteristics of pluripotent stem cells: they are capable of being grown in vitro without differentiating, they have a normal karyotype, and they remain capable of producing a number of different cell types. Clonally derived human embryonic stem cell lines maintain pluripotency and proliferative potential for prolonged periods in culture (Amit et al., Dev. Biol. 227:271, 2000). These cells hold considerable promise for use in human therapy, acting as a reservoir for regeneration of almost any tissue compromised by genetic abnormality, trauma, or a disease condition.
International Patent Publication WO 99/20741 (Geron Corp.) refers to methods and materials for growing primate-derived primordial stem cells. In one embodiment, a cell culture medium is provided for growing primate-derived primordial stem cells in a substantially undifferentiated state, having a low osmotic pressure and low endotoxin levels. The basic medium is combined with a nutrient serum effective to support the growth of primate-derived primordial stem cells and a substrate of feeder cells or an extracellular matrix component derived from feeder cells. The medium can further include non-essential amino acids, an anti-oxidant, and growth factors that are either nucleosides or a pyruvate salt.
A significant challenge to the use of stem cells for therapy is to control growth and differentiation into the particular type of tissue required for treatment of each patient.
U.S. Pat. No. 4,959,313 (M. Taketo, Jackson Labs) provides a particular enhancer sequence that causes expression of a flanking exogenous or recombinant gene from a promoter accompanying the gene that does not normally cause expression in undifferentiated cells. U.S. Pat. No. 5,639,618 (D. A. Gay, Plurion Inc.) proposes a method for isolating a lineage specific stem cell in vitro, in which a pluripotent embryonic stem cell is transfected with a construct in which a lineage-specific genetic element is operably linked to a reporter gene, culturing the cell under conditions where the cell differentiates, and then separation of cells expressing the reporter are separated from other cells.
U.S. Pat. No. 6,087,168 (Levesque et. al., Cedars Sinai Med. Ctr.) is directed to transdifferentiating epidermal cells into viable neurons useful for both cell therapy and gene therapy. Skin cells are transfected with a neurogenic transcription factor, and cultured in a medium containing an antisense oligonucleotide corresponding to a negative regulator of neuronal differentiation.
International Patent Publication WO 97/32025 (Mclvor et al., U. Minnesota) proposes a method for engrafting drug resistant hematopoietic stem cells. The cells in the graft are augmented by a drug resistance gene (such as methotrexate resistant dihydrofolate reductase), under control of a promoter functional in stem cells. The cells are administered into a mammal, which is then treated with the drug to increase engraftment of transgenic cells relative to nontransgenic cells.
International Patent Publication WO 98/39427 (Stein et al., U. Massachusetts) refers to methods for expressing exogenous genes in differentiated cells such as skeletal tissue. Stem cells (e.g., from bone marrow) are contacted with a nucleic acid in which the gene is linked to an element that controls expression in differentiated cells. Exemplary is the rat osteocalcin promoter. International Patent Publication WO 99/10535 (Liu et al., Yale U.) proposes a process for studying changes in gene expression in stem cells. A gene expression profile of a stem cell population is prepared, and then compared a gene expression profile of differentiated cells
International Patent Publication WO 99/19469 (Braetscher et al., Biotransplant) refers to a method for growing pluripotent embryonic stem cells from the pig. A selectable marker gene is inserted into the cells so as to be regulated by a control or promoter sequence in the ES cells, exemplified by the porcine OCT-4 promoter.
International Patent Publication WO 00/15764 (Smith et al., U. Edinburgh) refers to propagation and derivation of embryonic stem cells. The cells are cultured in the presence of a compound that selectively inhibits propagation or survival of cells other than ES cells by inhibiting a signaling pathway essential for the differentiated cells to propagate. Exemplary are compounds that inhibit SHP-2, MEK, or the ras/MAPK cascade.
Klug et al. (J. Clin. Invest. 98:216, 1996) propose a strategy for genetically selecting cardiomyocytes from differentiating mouse embryonic stem cells. A fusion gene consisting of the cc-cardiac myosin heavy chain promoter and a cDNA encoding aminoglycoside phosphotransferase was stably transfected into the ES cells. The resulting lines were differentiated in vitro and selected using G418. The selected cardiomyocyte cultures were reported to be highly differentiated. When engrafted back into mice, ES-derived cardiomyocyte grafts were detectable as long as 7 weeks after implantation.
Schuldiner et al. (Proc. Natl. Acad. Sci. USA 97:11307, 2000) report the effects of eight growth factors on the differentiation of cells from human embryonic stem cells. After initiating differentiation through embryoid body formation, the cells were cultured in the presence of bFGF, TGF-xcex21, activin-A, BMP-4, HGF, EGF, xcex2NGF, or retinoic acid. Each growth factor had a unique effect on the differentiation pathway, but none of the growth factors directed differentiation exclusively to one cell type.
There is a need for new approaches to generate populations of differentiated cells suitable for human administration.
This invention provides a system for depleting relatively undifferentiated cells from a heterogeneous cell population, such as may be obtained by differentiation of stem cells. The population is treated with a vector that puts a lethal or potentially lethal effector gene under control of a gene element that allows the gene to be expressed at a higher level in the undifferentiated subpopulation. This produces a population relatively enriched for mature cells, and suitable for use in regenerative medicine.
One embodiment of this invention is a population of cells differentiated from stem cells cultured ex vivo, which is essentially free of undifferentiated cells. Exemplary are pluripotent stem cells of primate origin, such as human embryonic stem cells.
Cells in the population can contain or be derived using a polynucleotide comprising the structure P-X, where X is a nucleic acid sequence that is lethal to a cell in which it is expressed, or renders a cell in which it is expressed susceptible to a lethal effect of an external agent; and P is a transcriptional control element that causes X to be preferentially expressed in undifferentiated cells. The connecting line in P-X indicates that the genetic elements are operatively linked, whether or not they are adjacent in the nucleic acid molecule.
X is referred to in the description that follows as an effector sequence. X can encode a toxin, a protein that induces or mediates apoptosis, or an enzyme (such as thymidine kinase) that converts a prodrug (such as ganciclovir) to a compound that is lethal to a cell in which X is expressed. Other examples are provided later in this disclosure.
In certain embodiments, P-X is an introduced heterologous molecule, meaning that the cell or its ancestors was genetically altered with a vector comprising P-X. In other embodiments the cell or its ancestors was genetically altered with a vector to place X under control of an endogenous transcriptional control element. Following transfection, X can be either transiently expressed in undifferentiated cells in the population, or P-X can be inheritable and expressed in undifferentiated progeny. Non-limiting examples for P include the OCT-4 promoter, and the promoter of telomerase reverse transcriptase (TERT). The cells can also contain a drug resistance gene Y under control of P, depicted in this disclosure as P-X-Y, indicating a functional relationship where P regulates transcription of both X and Y, with the elements being in any orientation in the sequence that links the functions in this manner.
Another embodiment of the invention is a stem cell genetically altered so as to contain a nucleic acid with the structure P-X, as already described. The invention also provides polynucleotide vectors adapted to genetically alter stem cells in this fashion.
Another embodiment of the invention is a method of producing a population of differentiated cells. A cell population comprising undifferentiated stem cells that contain a nucleic acid molecule comprising the structure P-X is treated to cause at least some undifferentiated cells in the population to differentiate.
Another embodiment of the invention is a method for depleting undifferentiated stem cells from a cell population. Stem cells in the population are genetically altered so that they contain a nucleic acid molecule comprising the structure P-X as already described. In this way, a gene that is lethal to a cell in which it is expressed, or renders it susceptible to a lethal effect of an external agent, is placed under control of a transcriptional control element that causes the gene to be preferentially expressed in undifferentiated cells. The cell population can be genetically altered when it is still predominantly undifferentiated (before being caused to differentiate), or when it already predominantly comprises differentiated cells.
If X is lethal to the cell, then undifferentiated stem cells can be depleted simply by culturing the cell population under conditions where X is expressed. If X renders the cell susceptible to lethal effects of an external agent (such as a drug or prodrug), then undifferentiated stem cells are depleted by combining the cells with the external agent. This can be done by contacting the cells with the agent in tissue culture, or administering the cells to the subject simultaneously or sequentially with the external agent, if not already present.
The reagents and techniques of this invention can be brought to bear on cell populations containing any type of stem cells. They are especially suited for application to primate pluripotent stem cells, such as human embryonic stem cells.
Other embodiments of the invention will be apparent from the description that follows.